Performance evaluation of the Roche Elecsys® Anti-SARS-CoV-2 immunoassays by comparison with neutralizing antibodies and clinical assessment

Quantitative measurement of SARS-CoV-2 neutralizing antibodies is highly expected to evaluate immune status, vaccine response, and antiviral therapy. The Elecsys® Anti-SARS-CoV-2 S (Elecsys® anti-S) was developed to measure anti-SARS-CoV-2 S proteins. We sought to investigate whether Elecsys® anti-S can be used to predict neutralizing activities in patients’ serums using an authentic virus neutralization assay. One hundred forty-six serum samples were obtained from 59 patients with COVID-19 at multiple time points. Of the 59 patients, 44 cases were included in Group M (mild 23, moderate 21) and produced 84 samples (mild 35, moderate 49), while 15 cases were included in Group S (severe 11, critical 4) and produced 62 samples (severe 43, critical 19). The neutralization assay detected 73% positive cases, and Elecsys® anti-S and Elecsys® Anti-SARS-CoV-2 (Elecsys® anti-N) showed 72% and 66% positive cases, respectively. A linear correlation between the Elecsys® anti-S assay and the neutralization assay were highly correlated (r = 0.7253, r2 = 0.5261) than a linear correlation between the Elecsys® anti-N and neutralization assay (r = 0.5824, r2 = 0.3392). The levels of Elecsys® anti-S antibody and neutralizing activities were significantly higher in Group S than in Group M after 6 weeks from onset of symptoms (p < 0.05). Conversely, the levels of Elecsys® anti-N were comparable in both groups. Three immunosuppressed patients, including cancer patients, showed low levels of anti-S and anti-N antibodies and neutralizing activities throughout the measurement period, indicating the need for careful follow-up. Our data indicate that Elecsys® anti-S can predict the neutralization antibodies in COVID-19.


Introduction
Coronavirus disease 2019 , caused by SARS-CoV-2, has been mainly diagnosed by reverse transcription-polymerase chain reaction (RT-PCR) that can directly detect the viral genomes [1]. Antigen testing has been also developed to detect pathogens rapidly without complicated procedures [2]. However, these tests cannot detect SARS-CoV-2 in certain periods after infection [3]. On the other hand, serological tests are essential tools to evaluate neutralizing antibody titers upon vaccination and to assess SARS-CoV-2 seroprevalence in cohorts [4,5]. Neutralizing antibodies targeting the receptor-binding domain (RBD) of the spike (S) protein can reduce viral infectivity by binding to the surface epitopes of viral particles which blocks virus entry [6]. Although the authentic virus neutralization assays can directly measure the neutralizing activities of the SARS-CoV-2 virus, those methods need to be performed in Biosafety Level 3 facilities, which limits their application [7]. Therefore, there is a need for safer, high-throughput, and widely available measurement methods that correlate well with neutralizing activities.
Recently, the Elecsys1 Anti-SARS-CoV-2 S (Elecsys1 anti-S) has been developed to quantitatively measure total antibodies against the S protein RBD, and the Elecsys1 Anti-SARS-CoV-2 (Elecsys1 anti-N) has been developed to semi-quantitatively measure total antibodies against SARS-CoV-2 N proteins that regulates viral replication [6] (Roche Diagnostics International Ltd, Rotkreuz, Switzerland). The aim of this study was to evaluate the feasibility and usefulness of these immunoassays by comparing the chronological seroprevalences in patients with various severity of COVID-19 along with the neutralizing activities measured by an authentic virus neutralization assay.

Clinical characteristics
This study complied with all relevant national regulations and institutional policies and was conducted in accordance with the tenets of the Declaration of Helsinki and was approved by the Institutional Review Board (IRB) at Juntendo University Hospital (IRB # 20-036). The need for informed consent from individual patients was waived because all samples were deidentified in line with the Declaration of Helsinki. From April to August 2020, 146 serum samples were collected from 59 patients with symptomatic COVID-19 confirmed by RT-PCR at multiple time points (number of samples per patient, median 2, IQR [2,3]). The periods between different time points were 2 to 58 days. Of the 59 COVID-19 patients, including 58 inpatients and one outpatient, 44 cases were included Group M (mild 23, moderate 21) and produced 84 samples (mild 35, moderate 49), while 15 cases were included Group S (severe 11, critical 4) and produced 62 samples (severe 43, critical 19). Clinical data were retrospectively collected from patients' charts. All samples were from the unvaccinated patients.
The results by Elecsys1 anti-S were quantitatively shown in units of U/mL with the cut-off point of 0.80 U/mL to differentiate samples as reactive (� 0.80 U/mL) and non-reactive (< 0.80 U/mL). The values between 0.40-250 U/mL represented a linear range, and the results below this range were set to 0.4 U/mL. The samples above 250 U/mL were diluted into the linear range of the assay (1:10 or 1:100) with Diluent Universal reagent (Roche Diagnostics, Rotkreuz, Switzerland). Thus, the applied setting enabled an upper limit of quantification of 25000 U/mL for these analyses before dilution. The Elecsys1 anti-N is a semi-quantitative assay, and the results were interpreted as follows: cutoff index (COI) <1.0 was non-reactive, and �1.0 was reactive.
Neutralization assay. The SARS-CoV-2 ancestral strain, WK-521 (lineage A, GISAID ID: EPI_ISL_408667), was used for the authentic virus neutralization assay that has been performed at the National Institute of Infectious Diseases (NIID) with ethics approval by the medical research ethics committee of NIID for the use of human subjects (#1178). Authentic virus neutralization assay has been performed as described previously [7]. Briefly, serially diluted serum samples (2-fold serial dilutions starting at 1:5 dilution, diluted with high glucose Dulbecco's Modified Eagle Medium supplemented with 2% Fetal Bovine Serum and 100 U/mL penicillin/streptomycin, from Fujifilm Wako Pure Chemicals, Japan) were mixed with the virus from 100 Median Tissue Culture Infectious Dose (TCID 50 ) and incubated at 37˚C for 1 hour. The mixture was subsequently incubated with VeroE6/TMPRSS2 cells (JCRB1819, JCRB Cell Bank, Japan) and seeded in 96-well flat-bottom plates for 4-6 days at 37˚C in a chamber supplied with 5% CO 2 . Then the cells were fixed with 20% formalin (Fujifilm Wako Pure Chemicals) and stained with crystal violet solution (Sigma-Aldrich, St Louis, MO). Each sample was assayed in 2-4 wells and the average cut-off dilution index of >50% cytopathic effect was presented as a neutralizing titer. Neutralizing titer of the sample below the detection limit (1:5 dilution) was set as 2.5. Neutralizing antibody titer of <5 is considered negative and >5 is considered positive.

Statistical analysis
Correlation studies were performed using Spearman's coefficient. Assay performance, linear regression, and curve fitting calculations were performed using Prism 9 (GraphPad Software, LLC, San Diego, CA, USA). For experiments involving two group comparisons, Wilcoxon signed-rank test was performed. The following notation was used to show statistical significance: � p value <0.05, �� p value <0.01, and ��� p value <0.001.

Results
As shown in Table 1, patients were classified into two groups according to the WHO criteria [WHO. Clinical management of COVID-19. Available from: https://www.who.int/ publications/i/item/clinical-management-of-covid-19]: Group M included mild and moderate cases and Group S included severe and critical cases.
We first compared the results of Elecsys1 anti-S and Elecsys1 anti-N to those of the authentic virus neutralizing assay. Of the 146 samples, the neutralization assay detected 73% (106/146) positives, and Elecsys1 anti-S and Elecsys1 anti-N showed 72% (105/146) and 66% (97/146) positives, respectively. These results were plotted, and the positive results were fitted with a linear regression. Fig 1A shows

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Evaluation of the Roche Elecsys Anti-SARS-CoV-2 immunoassays activities and antibodies tended to increase over time. However, there was no significant difference between Groups M and S until the sixth week. After the seventh week, the neutralization assay and Elecsys1 anti-S showed significantly higher values in Group S than in Group M (p < 0.05), which was not observed in the Elecsys1 anti-N results. Table 2 summarizes the details.
Since immunosuppressive therapies may cause false-negative results in antibody tests [9], the results after removing three patients receiving immunosuppressive therapies are shown in Fig 2D-2F. Fig 2D shows that the levels of neutralizing activities were higher in Group S than M after the fourth week (p < 0.05). Fig 2E shows that the levels of anti-S antibodies were significantly higher in Group S compared to Group M within the first 2 weeks and after the seventh week (p < 0.05). Fig 2F shows that the levels of anti-N antibodies were higher in Group S than M only in the first 2 weeks (p < 0.05).
Chronological changes in the results of the neutralization assay, Elecsys1 anti-S assay, and Elecsys1 anti-N assay were examined simultaneously in 23 inpatients who were tested in three time points or more. Table 3

Discussion
In this study, we demonstrated that the Elecsys1 anti-S assay detected anti-spike (S) protein RBD antibodies at about the same time that the virus neutralization assay detected neutralizing activity: 2 weeks after onset of symptoms. These results are consistent with a recent analysis of naturally acquired SARS-CoV-2 infected individuals showing that neutralizing antibodies were almost detectable about 14 days after infection [10]. A previous study has further reported that production of neutralizing antibodies within 14 days of onset of symptoms is an important factor in recovery [11]. In this study, we detected that there is a humoral increase in S-specific antibodies with neutralizing activity. The measurement values of Elecsys1 anti-S and neutralization assays were highly correlated, and the cases of Group S showed significantly higher values than Group M during the late phase of infection. The positive rate of the Elecsys1 anti-N assay was lower than that of the Elecsys1 anti-S and virus neutralization assays.
The measurement values of Elecsys1 anti-S and neutralization assays were highly correlated, and the cases of Group S showed significantly higher values than Group M during the late phase of infection. When patients receiving immunosuppressive treatment were removed from the cohort, both the Elecsys1 anti-S and Elecsys1 anti-N assays detected significantly higher levels of antibody in Group S than in Group M in the early stages of infection. It has been shown that patients undergoing immunosuppressive treatment have higher incidence rates and serious adverse events of COVID-19 [12]. Antibody levels in patients receiving chemotherapy, radiation therapy, and other immunosuppressive therapies require careful

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Evaluation of the Roche Elecsys Anti-SARS-CoV-2 immunoassays evaluation. It has been shown that IgG levels of SARS-CoV-2 S and N antibodies begin to decrease 2-3 months after infection [13,14] and neutralizing titers begin to decrease 8 months later [15][16][17]. Regarding the effects of plasmapheresis treatment, a previous case report demonstrated that S-protein IgG increased after plasma exchange within 3 days, and moderately decreased from day 3 to day 7, without any change in N-protein IgG [18]. In this study, however, no significant changes in antibody levels caused by plasmapheresis were observed. This study has several limitations: first, this study is a single-center study with limited sample size and short-term detections. Our results should be confirmed by additional assessments at other study sites. Second, variant determination has not been performed in the study. In terms of the SARS-CoV-2 strain that was used in the authentic virus neutralization assay, the utilized strain might have been different from the emerging variants. In Japan, lineage A and B variants were prevalent during the study period from April to August 2020 (1st and 2nd waves) [19]. We utilized the SARS-CoV-2 ancestral strain of lineage A for the authentic neutralization assay. On the other hand, the differences between the neutralizing antibody titers measured by the authentic neutralization assay and the commercially available immunoassays might depend on the viral variant, which is required to be verified in the future. Third, a comparably small sample size was utilized to measure agreement between the Elecsys1 anti-N immunoassays and a neutralization assay; as such, these data would benefit from further evaluation and validation. Forth, this study does not include samples from asymptomatic patients     nor from vaccinated patients. Therefore, the validity of our findings for patients with asymptomatic/mildly symptomatic SARS-CoV-2 infection after vaccination is yet to be shown and requires further study.
In conclusion, we found good correlation between SARS-CoV-2 viral S protein RBD levels measured by Elecsys1 anti-S and neutralizing antibody titers detected by an authentic virus neutralization assay. The findings of this study indicate that the quantitative detection of anti-SARS-CoV2 S protein by Elecsys1 anti-S assay reliably quantifies the antibody response to SARS-CoV-2, which is highly relevant to estimate protection.